Phylogenetic analysis and antimony resistance of Leishmania major isolated from humans and rodents.

Cutaneous Leishmaniasis (CL) is one of the world's neglected diseases which is caused by Leishmania spp. The aim of this study was to assess molecular profile and antimony resistance of Leishmania isolated from human and rodent hosts. Samples were collected from suspected CL patients referred to health centres and wild rodent's traps in Gonbad-e-Qabus region, north-eastern Iran. Smears were subjected to PCR-RFLP to identify Leishmania species. In addition, ITS1-PCR products were sequenced for phylogenetic analysis. Clinical isolates and rodent samples were subjected to MTT assay to determine IC50 values and in vitro susceptibilities. Expression levels of antimony resistance-related genes were determined in CL isolates. Out of 1,949 suspected patients with CL and 148 rodents, 1,704 (87.4%) and 6 (4.05%) were positive with direct smear, respectively. Digestion patterns of BusRI (HaeIII) endonuclease enzyme were similar to what expected for Leishmania major. Phylogenetic analysis revealed that the highest interspecies similarity was found between current L. major sequences with L. major obtained from Russia and Uzbekistan. Out of 20 L. major samples tested, 13 (65%) were resistant to meglumine antimoniate (MA) treatment, with an activity index (AI) exceeding 4. The remaining 7 samples (35%) responded to MA treatment and were classified as sensitive isolates, with a confirmed sensitive phenotype based on their AI values. The comparison expression analysis of three major antimony resistance-associated genes in unresponsive clinical isolates demonstrated significant fold changes for TDR1 (4.78-fold), AQP1 (1.3-fold), and γ-GCS (1.17-fold) genes (P < 0.05). Herein, we demonstrate genetic diversity and antimony resistance of L. major isolated from human and reservoir hosts in north-eastern Iran, which could be the basis for planning future control strategies.

Morphological and molecular characterization of Fasciola isolates from livestock in Golestan province, northern Iran.

BACKGROUND: Fascioliasis, caused by the liver flukes Fasciola hepatica and Fasciola gigantica, is a global zoonotic helminthic disease. The livestock and human are the final hosts of the parasites. Northern Iran is an important endemic region for fascioliasis. Few studies have been conducted on the characterization of Fasciola isolates from eastern regions of the Caspian littoral of the country. OBJECTIVE: The aim of the present study was to identify F. hepatica, F. gigantica and intermediate/hybrid forms of Fasciola isolates from livestock in Golestan province, northern Iran, using morphometric and molecular tools. METHODS: Livestock livers naturally infected with Fasciola spp. were collected from Golestan slaughterhouse during 2019-2020. The worms were morphometrically studied using a calibrated stereomicroscope. Genomic DNA was extracted from all samples, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on internal transcribed spacer (ITS1) region using Rsa1 restriction enzyme. All the isolates were then analysed by multiplex PCR on Pepck region. RESULTS: A total of 110 Fasciola isolates were collected from the infected livers, including 94 sheep, 12 cattle and 4 goats. Morphometric analysis of 61 adult Fasciola isolates indicated that, 44 and 17 isolates belonged to F. hepatica and F. gigantica, respectively. Eighty-one and 29 isolates belonged to F. hepatica and F. gigantica using ITS1-RFLP, respectively. However, Pepck Multiplex PCR indicated 72 F. hepatica, 26 F. gigantica and 12 intermediate/hybrid forms. All 12 hybrid isolates were found in sheep host. Two isolates were identified as F. gigantica using morphometry and F. hepatica using both molecular methods. CONCLUSION: The present study confirmed the existence of both F. hepatica and F. gigantica species and reported the first molecular evidence of hybrid Fasciola isolates in ruminants of Golestan province.

First molecular evidence of Theileria lestoquardi in small ruminants in northern Iran.

Ovine theileriosis as a critical agent in small ruminant production, can cause lethal infections. Different species of Theileria have been reported in various parts of the world, and each species causes different diseases in the host. This is the first molecular study to investigate the prevalence of ovine theileriosis and identify the dominant Theileria species in northern Iran. A number of 220 small ruminants, including sheep and goats, were randomly sampled from 22 flocks. Peripheral blood smears were stained by the Giemsa staining method. As well as for species identification, all samples were examined by PCR. From 220 samples, 160 and 60 were sheep and goat, respectively. By the Giemsa staining method, Theileria parasite was observed in 20 (9%) samples. But by polymerase chain reaction (PCR) method, 30 (13.6%) samples were positive for Theileria species. Theileria lestoquardi was the most common species found in these animals. The high prevalence of theileriosis in small ruminants demonstrates the emergence of ovine theileriosis in Mazandaran and Golestan provinces in northern Iran.

Domestic dogs carriers of Leishmania infantum, Leishmania tropica and Crithidia fasciculata as potential reservoirs for human visceral leishmaniasis in northeastern Iran.

BACKGROUND: In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%-80% of seropositive dogs are asymptomatic for Leishmania infection. OBJECTIVES: The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. METHODS: Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti-Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. RESULTS: Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody-positive dogs and 19 of the 25 (76%) PCR-positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR-RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR-positive samples. CONCLUSIONS: The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs.

Leishmania RNA virus 2 (LRV2) exacerbates dermal lesions caused by Leishmania major and comparatively unresponsive to meglumine antimoniate treatment.

PURPOSE: The present study investigated the possible role of Leishmania RNA virus 2 (LRV2) in the severity of dermal lesions and treatment failure due to Leishmania major. METHODS: The drug susceptibility of 14 clinical isolates of L.major, including resistant (n = 7) and sensitive (n = 7) isolates, was checked in the J774A.1 macrophage cell line. The presence of LRV2 among isolates was investigated by the RdRp gene and semi-nested PCR. Moreover, 1 × 106 sensitive L. major LRV2+ and LRV2- promastigotes were inoculated subcutaneously into the base tails of the 40 BALB/c mice divided into 4 groups (n = 10 in each group), including clinical LRV2+, clinical LRV2-, positive control LRV2+ and negative control LRV2-. The groups were infected with a unique isolate. The lesion size and parasite burden were evaluated. RESULTS: Sensitive and resistant isolates were determined by the drug susceptibility method. A higher presence of LRV2 was observed among MA-resistant isolates (6/7) compared with susceptible isolates (4/7), which was not statistically significant (P = 0.237). On the other hand, a comparison of the lesion sizes between the LRV2+ and LRV2- BALB/c mice groups revealed that the mean size of the lesion in the LRV2+ groups was significantly higher than the LRV2- (P = 0.034). In the same direction, there was an increased parasite burden in mice inoculated with LRV2+ groups compared with the LRV2- BALB/c mice groups (P = 0.002). CONCLUSIONS: Our findings showed that the presence of LRV2 could be one of the factors contributing to exacerbating CL. Although we found a higher presence of LRV2 in the resistant isolates, it seems that further investigations are recommended to determine the detailed association between lesions' aggravation and being comparatively unresponsive to treatment.

Expression and production of protoscolex recombinant P29 protein and its serological evaluation for diagnosis of human hydatidosis.

Cystic Echinococcosis/Hydatidosis considered as one of the most important parasitic diseases in humans and animals across the world. The goal of the present study was to determine a native antigen with an acceptable sensitivity and specificity to be used in the human hydatid cyst diagnostic methods. In the present study, recombinant P29 antigen was used to detect the antibodies in the serum of patients with hydatid cysts of the liver. In fact, purified recombinant P29 protein is used as an antigen in ELISA. In order to evaluate the recombinant P29 protein for diagnostic ELISA, 25 serums obtained from people harboring the hydatid cysts were tested. The result of the gene expression on a 12% SDS-PAGE gel showed a band with a length of 28 KD. Also, 28KDa band was observed through the reaction of recombinant P29 protein with Anti T7-tag monoclonal antibody in the western blotting method. This protein showed satisfactory results in detecting the hydatid cyst antibodies in the serum of patients having hydatid cysts. Twenty two of 25 hydatidosis serums positively reacted in ELISA using with P29 protein, indicating in 92% of ELISA sensitivity, 95% of specificity, 95.83% of positive predictive value, and 90.42% of negative predictive value for recombinant P29 protein. Whereas the produced recombinant protein P29 showed promising results in the diagnosis of hydatidosis but of more research needs to be done to reach a more accurate conclusion.

Global prevalence of Giardia duodenalis in cattle: A systematic review and meta-analysis.

Giardia duodenalis is an important intestinal parasite responsible for diarrhea in humans and animals worldwide. Up to now, G. duodenalis infections in cattle have been reported in many studies around the world. Hence, the aim of the present study is to report on the distribution of G. duodenalis in cattle at global scale and to evaluate the global prevalence, risk factors and genetic characterization of G. duodenalis infection among cattle worldwide. International databases were systematically searched to identify relevant studies. A random-effects meta-analysis model was used to estimate the overall and the subgroup-pooled prevalence of G. duodenalis across studies, and the variance between studies (heterogeneity) was quantified by I2 index. One hundred and fifty-eight articles (including 195 datasets), from 48 countries met eligibility criteria for analysis. Considering detection methods, the pooled prevalence was estimated to be 24% (95% confidence interval (CI), 19-30%) using copro-antigen techniques, 22% (95% CI, 17-28%) using molecular, and 16% (95% CI, 12-20%) using microscopic detection. Molecular methods showed that the highest number of reports were associated with assemblage E (45/46; 97.83% studies), assemblage A (33/46; 71.74% studies) and assemblage A+E (10/46; 21.74% studies). The pooled prevalence different of subgroups (WHO regions, countries, and type of cattle) were analyzed separately. Moreover, a significant association was observed between G. duodenalis infection with cattle suffering from diarrhea (odds ratio (OR), 2.61; 95% CI, 1.50-4.55) and pre-weaned calves (OR, 1.79; 95% CI, 1.08-2.95). These results suggest that the corresponding control scheme and effective management measures should be formulated to reduce the transmission of G. duodenalis infection according to the difference of geographical conditions in different areas.

Chromosome-scale Echinococcus granulosus (genotype G1) genome reveals the Eg95 gene family and conservation of the EG95-vaccine molecule.

Cystic echinococcosis is a socioeconomically important parasitic disease caused by the larval stage of the canid tapeworm Echinococcus granulosus, afflicting millions of humans and animals worldwide. The development of a vaccine (called EG95) has been the most notable translational advance in the fight against this disease in animals. However, almost nothing is known about the genomic organisation/location of the family of genes encoding EG95 and related molecules, the extent of their conservation or their functions. The lack of a complete reference genome for E. granulosus genotype G1 has been a major obstacle to addressing these areas. Here, we assembled a chromosomal-scale genome for this genotype by scaffolding to a high quality genome for the congener E. multilocularis, localised Eg95 gene family members in this genome, and evaluated the conservation of the EG95 vaccine molecule. These results have marked implications for future explorations of aspects such as developmentally-regulated gene transcription/expression (using replicate samples) for all E. granulosus stages; structural and functional roles of non-coding genome regions; molecular 'cross-talk' between oncosphere and the immune system; and defining the precise function(s) of EG95. Applied aspects should include developing improved tools for the diagnosis and chemotherapy of cystic echinococcosis of humans.

Role of neglected parasitic diseases in the era of COVID-19 pandemics.

There are some doubts about the exact relationship between neglected infectious diseases (NIDs) and COVID-19 disease, which remains to be clearly defined. The present review summarized the effect of parasitic infections as the risk factors or protective agents in the COVID-19 pandemic. Parasites could proficiently modulate immune responses. Thus, parasitic infections could have a different impact on the incidence and clinical severity of COVID-19 in different regions of the world. Also, restoring programs to prevent, treat, and control NIDs, in particular helminths, could help in reducing the incidence and mortality of COVID-19 in endemic areas and help to increase vaccination effectiveness. Changes in the gut microbiome associated with helminth infection may have systemic immunomodulatory effects toward suppressing host immune responses, reducing vaccine efficacy and increasing the severity of other infectious diseases. The cytokine storm observed in severe cases of COVID-19 is characterized by a predominance of proinflammatory cytokines, such as IL-6. However, it is possible that helminth infection could change the outcome of infection by modifying the Th2 response to limit the inflammatory component; this would be particularly apparent in areas endemic for helminthic infections, which suggests a possible protective effect against COVID-19. Because parasitic infections affect more than 2 billion people throughout the world, their impact on COVID-19-associated effects on public health could be considerable. Further studies with larger sample sizes would be needed to explore the possible role of neglected parasitic infections in the COVID-19 pandemic.

Corynosoma caspicum (Acanthocephala, Polymorphidae), as a heavy metal bioindicator in the fish Gasterosteus aculeatus from the Caspian Sea, northern Iran.

Previous marine biology studies found that the concentration of heavy metals in some parasites of fish such as acanthocephalans can be a proper bioindicator. Therefore, we attempted to measure five heavy metal concentrations in the tissues of the fish Gasterosteus aculeatus (G. aculeatus) and its acanthocephalan parasites, Corynosoma caspicum (C. caspicum) from the Southern Caspian Sea, northern Iran. G. aculeatus (three-spined stickleback) was collected from the south of the Caspian Sea, Mazandaran Province, northern Iran. After tissue preparation, the heavy metal concentrations in fishes and acanthocephalans were obtained using the tissue dissolution technique and an atomic absorption spectrophotometer. The concentrations of chromium (Cr), cadmium (Cd), lead (Pb), zinc (Zn), and copper (Cu) in the skin, liver, muscle, and intestine tissues of the fish and its parasites, C. caspicum, were measured and compared. Eighty (32%) of 250 collected fish were infected by at least one acanthocephalan parasite. The Cr indicated the highest concentration (5.329±3.275) of the heavy metals in acanthocephalan, even more than the skin, liver, and muscle of infected fishes. Cd had the lowest concentration (0.0333±0.0075) of heavy metals in acanthocephalan, but it was still higher than the concentration in the infected fishes' skin, liver, muscle, and intestine tissues. Our findings indicated that C. caspicum parasites can be considered extremely sensitive early-alert bioindicators, particularly in sensitive and under-threat environments with low pollution levels.

Seroprevalence, risk factors, and clinical symptoms of Toxocara spp. infection among children 3-15 years old in northern Iran.

The World Health Organization has categorized toxocarosis as a neglected tropical disease despite its significant impact on high-risk groups such as children. This study aimed to investigate the seroprevalence, risk factors, and clinical symptoms of Toxocara spp. infection among children 3-15 years old in northern Iran. A total of 386 children were enrolled in the study. All serum samples were tested for the presence of IgG antibodies against Toxocara spp. infection using an enzyme-linked immunosorbent assay. Moreover, relevant risk factors and clinical symptom data were obtained using questionnaires. Data analysis was performed using the SPSS software version 24. The overall seroprevalence of Toxocara spp. infection was found 2.85 % (11/386). However, Toxocara spp. infection was high for some risk factors, including eating soil (14.3 %), contacting cats (6.7 %), and consuming raw vegetables (3.7 %). However, there were no statistically significant differences regarding the risk factors and socio-demographic characteristics. Considering the clinical symptoms, Toxocara spp. infection was different in children with eosinophilia (20 %), ocular disorders (8.3 %), skin disorders (7.7 %), liver disorders (4.5 %), and stomach ache (4.2 %), although not statistically significant. The results revealed that the seroprevalence of Toxocara spp. infection was relatively low in children in northern Iran. It is suggested to conduct more studies in different parts of Iran to gain a deeper understanding of the toxocarosis seroprevalence and its status in high-risk groups such as children with asthma, hypereosinophilic syndrome, allergic skin disorders, and epilepsy.

Global prevalence of microsporidia infection in cats: A systematic review and meta-analysis of an emerging zoonotic pathogen.

Microsporidiosis in pet and stray cats is an emerging zoonotic threat with public health significance worldwide. However, the epidemiological patterns of feline microsporidiosis is still neglected around the world. Hence, current systematic review and meta-analysis aimed at characterizing the prevalence estimates and genotypes of microsporidian parasites among cats of the world. Several databases (PubMed, Web of Science, Scopus, and Google scholar) were systematically explored to find relevant studies. Evaluation of the weighted prevalences among included studies was done using random-effects model. Totally, 30 studies (34 datasets) reported from 19 countries were included in the present work. Microsporidia infection demonstrated higher prevalence rates using microscopy 29.7 % (19.7-42.2 %), followed by serology and molecular techniques with 11 % (4.6-24.2 %) and 8.2 % (5.9-11.4 %), respectively. Moreover, molecular data showed Enterocytozoon bieneusi as the most dominant reported species with 7.4 % (5.1-10.5 %). Also, investigations (11 studies) mostly isolated D genotype among all E. bieneusi genotypes. These results highlight cats as a potential reservoir for acquisition of microsporidia infection in humans, and surveillance programs should be implemented in high-risk areas.

Serological Study of Fascioliasis Using Indirect ELISA in Gorgan City, Golestan Province, Northern Iran.

BACKGROUND: Fascioliasis is a neglected zoonotic disease, caused by Fasciola species in human and livestock. We aimed to detect the seroprevalence of human fascioliasis Gorgan City, Golestan Province, northern Iran using ELISA method in 2017. METHODS: Overall, 612 serum samples were analyzed. A relevant questionnaire for demographic data was obtained for all cases. An indirect ELISA test was used to detect IgG antibodies against Fasciola in the sera. The data analysis was performed employing SPSS program version 21. RESULTS: Eleven cases (1.79%) were seropositive for fascioliasis. The seroprevalence of fascioliasis was 1.9% and 1.1% among males and females, respectively. There was no statistically significant association between the fascioliasis and analyzed variables such as sex, age, residence, job, education, etc. CONCLUSION: This study was conducted only on the people referring to the Reference Laboratory of Gorgan. It cannot be distributed to the whole city. Thus, due to importance of the disease, finding the seroprevalence of fascioliasis in a comprehensive survey in Golestan Province should be accounted in further studies.

Echinococcus multilocularis and Echinococcus granulosus in canines in North-Khorasan Province, northeastern Iran, identified using morphology and genetic characterization of mitochondrial DNA.

BACKGROUND: Canids are definitive hosts of Echinococcus multilocularis and Echinococcus granulosus. This study aimed to survey these two Echinococcus species in canids of North-Khorasan Province, northeastern Iran, using morphological criteria and genetic characterization of mitochondrial DNA. METHODS: The carcasses of 106 canids, namely 61 jackals (Canis aureus), 23 foxes (Vulpes vulpes), 19 dogs (Canis familiaris) and three wolves (Canis lupus) were collected from the study area in 2013-2014 and examined for Echinococcus species. Morphological features were assessed by microscopy of adult worms. For molecular characterization, DNA was extracted, mostly from the adult worms but also from eggs. DNA fragments of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) mitochondrial genes were amplified and sequenced. Sequences were aligned and compared with reference sequences. Intraspecific and interspecific diversity were calculated and phylogenetic analysis was performed. RESULTS: Overall, 9.4% of the canids (eight jackals and two foxes) were found infected with E. multilocularis by molecular methods, of which seven cases were also confirmed using morphological description of the adult worms. Echinococcus granulosus was found in 6.6% of the canines (four dogs, two jackals and one wolf) as determined by both molecular methods and adult cestode morphology. All E. granulosus isolates were identified as the G1 genotype. Comparative sequence analysis indicated 0-0.7% and 0% intraspecific divergence within E. granulosus isolates and 0% and 0-0.2% within E. multilocularis isolates for cox1 and nad1, respectively. CONCLUSIONS: This study revealed the presence of E. multilocularis and E. granulosus in canids of North-Khorasan Province of Iran. Jackals were found infected with both E. multilocularis and E. granulosus, but infection with the former species was higher.

Seroprevalence of Hydatidosis in People Referring to Reference Laboratory of Gorgan, Golestan Province, Northern Iran 2017.

BACKGROUND: Hydatidosis is a neglected global zoonotic disease, caused by larval stage of the cestode Echinococcus granulosus in human and animal. Because of high economic and medical importance of the disease, this study was performed to find the seroprevalence of human hydatidosis in Gorgan City, Golestan Province, northern Iran. METHODS: In this cross-sectional study, blood samples were collected from people referring to Reference laboratory of Golestan University of Medical sciences in 2017. A relevant questionnaire was completed for demographic data for each person. Echinococcus IgG antibody was investigated by ELISA using native antigen B. The data were analyzed using SPSS software applying logistic regression. RESULTS: Overall, 612 blood samples were collected. Cut-off was considered 0.29. Sixteen cases (2.6%) were seropositive for hydatidosis. The seroprevalence of hydatidosis was 2.3% and 4.7% among males and females, respectively. There was no statistically significant correlation between the hydatidosis and investigated variables such as sex, age, tribes, residence, education, etc. CONCLUSION: The prevalence of human hydatidosis shows approximately the same range as other regions of Iran. Although due to the neighboring the Mazandaran Province reported as the highest seroprevalence of hydatidosis, we expected more rate of seropositivity.

Analysis of nad2 and nad5 enables reliable identification of genotypes G6 and G7 within the species complex Echinococcus granulosus sensu lato.

The larval stages of tapeworms in the species complex Echinococcus granulosus sensu lato cause a zoonotic disease known as cystic echinococcosis (CE). Within this species complex, genotypes G6 and G7 are among the most common genotypes associated with human CE cases worldwide. However, our understanding of ecology, biology and epidemiology of G6 and G7 is still limited. An essential first step towards this goal is correct genotype identification, but distinguishing genotypes G6 and G7 has been challenging. A recent analysis based on complete mitogenome data revealed that the conventional sequencing of the cox1 (366 bp) gene fragment mistakenly classified a subset of G7 samples as G6. On the other hand, sequencing complete mitogenomes is not practical if only genotype or haplogroup identification is needed. Therefore, a simpler and less costly method is required to distinguish genotypes G6 and G7. We compared 93 complete mitogenomes of G6 and G7 from a wide geographical range and demonstrate that a combination of nad2 (714 bp) and nad5 (680 bp) gene fragments would be the best option to distinguish G6 and G7. Moreover, this method allows assignment of G7 samples into haplogroups G7a and G7b. However, due to very high genetic variability of G6 and G7, we suggest to construct a phylogenetic network based on the nad2 and nad5 sequences in order to be absolutely sure in genotype assignment. For this we provide a reference dataset of 93 concatenated nad2 and nad5 sequences (1394 bp in total) containing representatives of G6 and G7 (and haplogroups G7a and G7b), which can be used for the reconstruction of phylogenetic networks.

Genetic and morphometric categorization of Taenia ovis from Sheep in Iran.

Little is known about the genetic and morphological characters of Taenia ovis. The purpose of the present study was to characterize sheep isolates of T. ovis using rostellar hook morphometry as well as mitochondrial genes sequence analysis. Ninety sheep specimens of Cysticercus ovis were collected from 18 slaughterhouses in Iran. The mean ± s.d. for total length of large and small hooks were 174.1 ± 6.4 and 116.7 ± 5.4 µm, respectively. CO1 and 12S rRNA sequence analysis showed 11 and nine haplotypes, respectively. The level of pairwise nucleotide variations between individual haplotypes of CO1 and 12S rRNA genes were 0.3-1.1 and 0.2-1.0%, respectively. Level of nucleotide variation in CO1 and 12S rRNA between T. ovis haplotypes from present study and eight other Taenia species was found to be 11.3-17.8 and 5.3-16.3%, respectively. Phylogenetic analysis clustered all T. ovis isolates into a single clade comprised of the all CO1 and 12S rRNA haplotypes. CO1 nucleotide difference between T. ovis ovis and T. asiatica was 13.6% that is lesser than the corresponding difference between T. ovis ovis and T. ovis krabbei, warranting the designation of two separate species as T. ovis and T. krabbei. Interclass correlation coefficients showed that there was no significant association between rostellar hook length variation and the variability of the mitochondrial genes.

The benefits of analysing complete mitochondrial genomes: Deep insights into the phylogeny and population structure of Echinococcus granulosus sensu lato genotypes G6 and G7.

Cystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of the species complex Echinococcus granulosus sensu lato. Within this complex, genotypes G6 and G7 have been frequently associated with human CE worldwide. Previous studies exploring the genetic variability and phylogeography of genotypes G6 and G7 have been based on relatively short mtDNA sequences, and the resolution of these studies has often been low. Moreover, using short sequences, the distinction between G6 and G7 has in some cases remained challenging. The aim here was to sequence complete mitochondrial genomes (mitogenomes) to obtain deeper insight into the genetic diversity, phylogeny and population structure of genotypes G6 and G7. We sequenced complete mitogenomes of 94 samples collected from 15 different countries worldwide. The results demonstrated that (i) genotypes G6 and G7 can be clearly distinguished when mitogenome sequences are used; (ii) G7 is represented by two major haplogroups, G7a and G7b, the latter being specific to islands of Corsica and Sardinia; (iii) intensive animal trade, but also geographical isolation, have likely had the largest impact on shaping the genetic structure and distribution of genotypes G6 and G7. In addition, we found phylogenetically highly divergent haplotype from Mongolia (Gmon), which had a higher affinity to G6.

Distinguishing Echinococcus granulosus sensu stricto genotypes G1 and G3 with confidence: A practical guide.

Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing the differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1.